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Gutell 088.mpe.2003.29.0258


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Gutell 088.mpe.2003.29.0258

  1. 1. Phylogenetic relationships among 28 spirotrichous ciliatesdocumented by rDNAElizabeth A. Hewitt,aKirsten M. M€uuller,bJamie Cannone,cDaniel J. Hogan,aRobin Gutell,cand David M. Prescotta,*aDepartment of Molecular, Cellular and Developmental Biology, University of Colorado, Boulder, CO 80309-0347, USAbDepartment of Biology, University of Waterloo, Waterloo, Ont., Canada N2L 3G1cInstitute for Cellular and Molecular Biology and Section of Integrative Biology, University of Texas at Austin, Austin, TX 78712-1095, USAReceived 23 August 2002; revised 5 March 2003AbstractThe contiguous sequence of the SSU rDNA, ITS 1, 5.8S, ITS 2, and 1370 bp at the 50end of the LSU rDNA was determined in25 stichotrichs, one oligotrich, and two hypotrichs. Maximum parsimony, neighbor-joining, and quartet-puzzling analyses wereused to construct individual phylogenetic trees for SSU rDNA, for LSU rDNA, and ITS 1 + 5.8S + ITS 2, as well as for all thesecomponents combined. All trees were similar, with the greatest resolution obtained with the combined components. Phylogeneticrelationships were largely consistent with classical taxonomy, with notable disagreements. DNA sequences indicate that Oxytrichagranulifera and Oxytricha longa are rather distantly related. The oligotrich, Halteria grandinella, is placed well within the orderStichotrichida. Uroleptus pisces and Uroleptus gallina probably belong to different genera. Holosticha polystylata (family Holosti-chidae) and Urostyla grandis (family Urostylidae) are rather closely related. These rDNA sequence analyses imply the need for somemodifications of classical taxonomic schemes.Ó 2003 Elsevier Science (USA). All rights reserved.1. IntroductionSpirotrichs are a particularly interesting group ofciliates because of complex evolutionary modificationsof their micronuclear (germline) and macronuclear (so-matic) genomes and because of the extraordinary ma-nipulations of DNA required to convert a micronucleargenome into a macronuclear genome after cell mating(Prescott, 2000). Micronuclear genes are interrupted byshort, noncoding, AT-rich segments called internaleliminated segments, or IESs, first discovered in spiro-trichs (Klobutcher et al., 1984) and subsequently iden-tified in micronuclear genes in Paramecium (Steele et al.,1994). IESs divide a gene into segments called macro-nuclear destined segments, or MDSs. During macro-nuclear development IESs are spliced out of the DNA,and MDSs are ligated to form transcriptionally com-petent genes. In some micronuclear genes in stichotrichsrecombination between IESs within the gene has oc-curred in the course of evolution, causing MDSs tobecome disordered, or scrambled (Prescott, 2000).Scrambled MDSs become unscrambled and ligated inthe orthodox order during macronuclear development.Numbers, sizes, sequences, and positions of IESswithin a particular micronuclear gene differ from onestichotrich to another, reflecting both a high rate in theaccumulation of mutations in IESs (DuBois and Pres-cott, 1997) and the ability of IESs to migrate alongDNA (DuBois and Prescott, 1995). The IES differences,in turn, generate differences from organism to organismin the numbers, sizes and patterns of MDSs within aparticular micronuclear gene. IES/MDS patterns in amicronuclear gene in a series of stichotrichs instruct usabout the origin and evolution of IESs and MDSs whenthey are placed in the context of the phylogenetic rela-tionships among those organisms (Hogan et al., 2001).Phylogenetic relationships among stichotrichs have tra-ditionally relied on morphological features, particularlyMolecular Phylogenetics and Evolution 29 (2003) 258–*Corresponding author. Fax: 1-303-492-7744.E-mail address: (D.M. Prescott).1055-7903/$ - see front matter Ó 2003 Elsevier Science (USA). All rights reserved.doi:10.1016/S1055-7903(03)00097-6
  2. 2. on the numbers, patterns, and positions of cirri (kin-eties) and membranelles. These characteristics are notinherited through nuclear genes and are thus qualitativeand, at best, semiquantitative features that do not ade-quately define phylogenetic relationships. Therefore, wehave determined nuclear rDNA sequences for 25 sti-chotrichs, one oligotrich, and two hypotrichs, in whichwe are currently studying the structure of several mi-cronuclear genes, in order to document phylogeneticrelationships more precisely. We can then use these re-lationships to elucidate the origin and evolution of IESsand MDS scrambling. Phylogenetic relationships docu-mented by nuclear rDNA will also help in the appro-priate classification of spirotrichs, includingunidentified/unnamed spirotrichs, four examples ofwhich are present among the 28 organisms used in thepresent study. Finally, classifications of spirotrichs thathave been based on morphological criteria may needsome adjustment when nuclear rDNA sequences areavailable to define phylogenetic relationships.2. Materials and methods2.1. Origin of organisms1. Gastrostyla steineii. A gift from W. Foissner.2. Sterkiella nova (formerly Oxytricha nova; Foissnerand Berger, 1999). Isolated from a pond in Burling-ton, North Carolina.3. Sterkiella sp. (Aspen). Isolated from the RoaringFork River, Aspen, Colorado.4. Pleurotricha lanceolata. Isolated from Ten MileCreek, Colorado. Identified by W. Foissner.5. Unknown F. Isolated from Ten Mile Creek, Colo-rado.6. Tetmemena pustulata. Isolated from a pond on theUniversity of Colorado campus, Boulder, Colorado.Identified by W. Foissner.7. Stylonychia sp. (Aspen). Isolated from Ten MileCreek, Colorado.8. Oxytricha sp. (Misty). Isolated from Misty CreekPond, Sarasota, Florida.9. Oxytricha longa. Isolated from Ten Mile Creek,Colorado. Identified by W. Foissner.10. Unknown B. Isolated from the Roaring Fork River,Colorado.11. Cyrtohymena citrina. Isolated from Maroon Creek,Aspen, Colorado. Identified by W. Foissner.12. Paraurostyla weissei. Isolated from Teller Lake,Boulder County, Colorado. Identified by W. Foiss-ner.13. Stylonychia lemnae. Isolated from Teller Lake,Boulder County, Colorado.14. Stylonychia mytilus. Isolated in Harbin, China.Identification confirmed by W. Foissner.15. Paruroleptus lepisma. Isolated from Ten Mile Creek,Colorado. Identified by W. Foissner.16. Uroleptus gallina. Isolated from Teller Lake, Boul-der County, Colorado.17. Uroleptus pisces. Isolated from Teller Lake, BoulderCounty, Colorado.18. Urostyla grandis. Isolated from a pond on the Uni-versity of Colorado campus, Boulder, Colorado.19. Holosticha polystylata. Isolated from a pond on theUniversity of Colorado campus, Boulder, Colo-rado.20. Oxytricha granulifera. Isolated from the RoaringFork River, Aspen, Colorado. Identified byW. Foissner.21. Unknown SHS. Isolated from a hot spring (43 °C) inSteamboat Springs, Colorado.22. Paraurostyla viridis. Isolated from Misty CreekPond, Sarasota, Florida.23. Halteria grandinella. Isolated from a pond on theUniversity of Colorado campus, Boulder, Colorado.24. Engelmanniella mobilis. A gift from W. Foissner25. Moneuplotes crassus. Macronuclear DNA was a giftfrom Carolyn Price, University of Cincinnati.26. Euplotes aediculatus. Isolated from Teller Lake,Boulder County, Colorado.27. Sterkiella histriomuscorum (formerly named Oxytri-cha trifallax). Isolated from the Jordan River,Bloomington, Indiana.28. Unknown FL. Isolated from Misty Creek Pond,Sarasota, Florida.Most organisms were cultured on Chlorogoniumelongata in open dishes with the following exceptions.Engelmanniella mobilis was cultured on a mixture ofunidentified bacteria. Urostyla grandis was cultured onTetrahymena thermophila. Halteria grandinella was cul-tured monaxenically on Chlorogonium. Moneuplotescrassus was not cultured; macronuclear DNA was a giftof Carolyn Price.Macronuclei were purified from 10 to 30 packed ml ofcells by the method described previously (Prescott andGreslin, 1992).Polymerase chain reactions (PCR) were performedwith macronuclear DNA using terminal SSU rDNAuniversal primers (Elwood et al., 1985). Amplified DNAwas electrophoretically analyzed in a 1% agarose gel tocheck for appropriate size products. PCR products werepurified (Qiagen, Chatsworth, CA) and directly se-quenced using internal primers (Elwood et al., 1985) inthe MCDB Departmental sequencing facility.Sequences of ITS 1, 5.8S, ITS 2 and 1370 bp of thelarge subunit rDNA were obtained by TA cloning ofPCR products using either pGEM easy T vector (Pro-mega, Madison, WI) or pKRX. PCR primers weredesigned from SSU sequences of Sterkiella histriomus-corum and from LSU sequence of Euplotes aediculatusE.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267 259
  3. 3. (M98383 and M98377). Plasmid clones containinginserts of the appropriate size were prepared using theQia prep spin miniprep kit (Qiagen, Chatsworth, CA).Sequencing was performed in the MCDB departmentalsequencing facility using M13 forward and reverseprimers.2.2. Alignment of nuclear rDNA genesThe nuclear rDNA gene sequences were alignedmanually with the SUN microsystems Unix-basedalignment editor AE2 (developed by T. Macke, seeLarsen et al., 1993). Sequences were juxtaposed in thealignment to represent, as best as possible, the similarplacement of homologous nucleotides in two and threedimensional space. Sections of the rRNA with signifi-cant amounts of sequence identity can be aligned solelywith the sequence information. However, regions withsignificant amounts of sequence variation require sec-ondary structure information to align the sequencesaccurately and confidently. Covariation analysis hasbeen used to accurately predict rRNA secondarystructure and the beginnings of its tertiary structure(Gutell et al., 1985, 2002). The secondary structuremodels for a large phylogenetically diverse collection ofSSU and LSU rRNAs is available at the ComparativeRNA Web site (, Can-none et al., 2002). Usually these variable regions con-tain a structure that is conserved in the differentsequences, and thus nucleotides can be aligned withabsolute, or near absolute confidence. We are confidentin the alignment of the entire SSU, LSU, and 5.8SrRNAs.2.3. Phylogenetic analysis of nuclear rDNA genesMaximum parsimony, neighbor-joining and quartet-puzzling analyses were performed using PAUP 4.0 beta10 (Phylogenetic Analysis Using Parsimony; Swofford,2002). Parsimony analysis was applied using heuristicsearches under the conditions of random addition oftaxa (100 replicates), steepest descent, tree bisection-re-connection (TBR) branch swapping and the MUL-PARS option. The quartet-puzzling method appliesmaximum-likelihood tree reconstruction to all possiblequartets that can be formed from all sequences thatserve as starting points to reconstruct a set of optimaln-taxon trees (Strimmer and von Haeseler, 1996). Thismethod has been shown to be equivalent or better forreconstructing the true tree than neighbor-joiningmethods (Strimmer and von Haeseler, 1996). The valuesrepresent the percentage of times that a particularcluster was found among the 1000 intermediate trees(QPS values). Neighbor-joining analyses were con-ducted on a matrix of distance values estimated ac-cording to the Kimura 2-parameter model (Kimura,1980) using a transition/ transversion ratio of 2.0 and asingle-category substitution rate. Support for nodes onparsimony and distance trees was established usingbootstrap resampling (1000 replicates). The nuclearrDNA sequence from T. thermophila (GenBank Acces-sion No. X54512) was used as an outgroup taxon for allanalyses of the nuclear rDNA genes. Tetrahymena isdistantly related to spirotrichs based on a variety oftaxonomic/genetic criteria.3. Results and discussionSequence data for all taxa included in this study weresubmitted to GenBank and accession numbers are givenin Table 1.3.1. Phylogenetic analysis of nuclear SSU rDNA genesThe nuclear SSU rDNA genes were amplified byPCR and sequenced for all taxa included in the presentstudy. Parsimony analysis of 331 phylogenetically in-formative characters of the macronuclear SSU rDNAgene resulted in 180 most parsimonious trees with alength of 855 and a consistency index (CI) of 0.595. Astrict consensus of these 180 most parsimonious trees isdepicted in Fig. 1. The most parsimonious trees differ intopology because of unresolved nodes and brancheswith less than 50% support, as seen in Fig. 1. Treesgenerated from both neighbor-joining and maximumlikelihood quartet-puzzling analyses show similar to-pologies and hence only the maximum parsimony tree isshown. However, bootstrap values for the neighbor-joining and quartet-puzzling analyses are shown on thetree (see Fig. 1 legend). Resolution is low among thevarious clades seen in Fig. 1, and the species in somegenera do not appear to form monophyletic groups(e.g., Sterkiella, Oxytricha, and Paraurostyla, Fig. 1).Some groups are well supported, for example, the groupcontaining Stylonychia mytilus, Stylonychia lemnae, andStylonychia sp. Aspen is strongly supported by parsi-mony (95%) and neighbor-joining bootstrap analyses(97%) though not well supported by quartet-puzzlinganalysis (62%). Among unidentified ciliates, Unknown Fis clearly associated with Pleurotricha lanceolata, andthis relationship is well supported [97% parsimony (MP)bootstrap support, 93% quartet-puzzling steps (QPS),and 87% neighbor-joining (NJ) bootstrap support], as isUnknown SHS with Oxytricha longa [98% (MP), 89%(QP), and 100% (NJ)]. However, Unknown FL is notclearly associated with any particular ciliate but ismoderately supported [89% (MP), 87% (QPS), and 91%(NJ)] within a clade containing taxa from a number ofgenera (Stylonychia, Oxytricha, Gastrostyla, Tetmem-ena, and Pleurotricha) (Fig. 1). Most interesting is thatO. longa and Oxytricha granulifera do not group to-260 E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267
  4. 4. gether in any of the molecular trees, as would be ex-pected based on classical taxonomy. In fact, these twotaxa differ from each other by 3.39% (corrected sequencedivergence).The corrected sequence divergence of the macronu-clear SSU rDNA gene among the various ciliates rangedfrom 0.51% (P. lanceolata and Unknown F) to as highas 22.9% (T. thermophila and M. crassus). E. aediculatusand M. crassus are well supported as a solid entity in allphylogenetic trees (see bootstrap values in Fig. 1) andare positioned on a long branch in the neighbor-joiningtree (not shown). They differ considerably in sequencedivergence from the remaining ciliates (not includingoutgroup taxon), ranging from 15.7% (M. crassus andUnknown F) to 16.6% (M. crassus and H. grandinella).In terms of the unknown taxa, the sequence divergenceis low between Unknown SHS and O. longa (0.91%),and between Unknown F and P. lanceolata (0.51%). Onthe other hand, Unknown FL, which is moderatelysupported as grouping with a number of genera, differsfrom these taxa ranging from 1.58% (Oxytricha sp.Misty) to 2.49% (Tetmemena pustulata). Similarly, Un-known B, which is moderately supported as groupingwith Cyrtohymena citrina and Paraurostyla weissei dif-fers from these taxa by 1.75 and 1.41%, respectively.3.2. Phylogenetic analysis of nuclear LSU rDNA genesApproximately 1370 bp at the 50end of the nuclearLSU rDNA genes were amplified by PCR and se-quenced. Parsimony analysis of 462 phylogeneticallyinformative characters of the nuclear LSU rDNA genesresulted in 38 most parsimonious trees with a length of786 and a consistency index (CI) of 0.495 and for whicha strict consensus is depicted in Fig. 2. The most parsi-monious trees differed in topology due to unresolvednodes and branches with 50% bootstrap values. Treesgenerated from both neighbor-joining and maximumlikelihood quartet-puzzling analyses showed similar to-pologies, and hence only the maximum parsimony tree isshown. Similar to the macronuclear SSU analysis, thereis low resolution among many clades, and several of thegenera also do not form monophyletic clades (Oxytri-cha, Sterkiella, Uroleptus, Paraurostyla, etc.). In fact, thetopology of the phylogenetic tree in Fig. 2 is nearlyidentical to that of the macronuclear SSU rDNA treedepicted in Fig. 1. The unknown taxa in this analysisform similar relationships to those seen in Fig. 1 withthe exception that Unknown B is not associated withC. citrina and P. weissei, and relationships of thisunknown to other ciliates are poorly understood.Table 1Lengths (bp) of the SSU rDNA, ITS 1, 5.8S, ITS 2 and LSU rDNA sequenced in 28 Spirotrichs and Tetrahymena thermophilaSSU ITS 1 5.8S ITS 2 26S Accession Nos.Unknown F 1771 127 153 203 1368 AF508777Pleurotricha lanceolata 1771 127 153 203 1369 AF508768Tetmemena pustulata 1771 119 153 203 1367 AF508775Oxytricha sp. (Misty) 1771 131 153 203 1369 AF508764Sterkiella nova 1771 131 153 204 1370 AF508771Gastrostyla stenii 1771 131 153 192 1369 AF508758Sterkiella sp. (Aspen) 1771 132 153 203 1370 AF508772Sterkiella histriomuscorum 1771 130 153 203 1369 AF508770Unknown FL 1771 124 153 203 1369 AF508778Stylonychia sp. (Aspen) 1769 132 153 203 1367 AF508754Stylonychia mytilus 1771 124 153 203 1367 AF508774Stylonychia lemnae 1770 121 153 203 1368 AF508773Paraurostyla weissei 1771 121 153 203 1366 AF508767Cyrtohymena citrina 1772 134 153 204 1367 AF508755Unknown B 1770 132 153 204 1369 AF508776Unknown SHS 1774 127 153 201 1371 AF508769Oxytricha longa 1770 128 153 201 1369 AF508763Oxytricha granulifera 1774 124 153 198 1365 AF508762Paraurostyla viridis 1774 124 153 198 1365 AF508766Engelmanniella mobilis 1773 121 154 201 1375 AF508757Halteria grandinella 1779 125 153 194 1370 AF508759Uroleptus gallina 1775 129 153 192 1369 AF508779Paruroleptus lepisma 1772 125 153 192 1365 AF508765Uroleptus pisces 1772 130 153 200 1327 AF508780Holosticha polystylata 1769 128 153 192 1356 AF508760Urostyla grandis 1768 130 153 198 1366 AF508781Moneuplotes crassus 1890 113 150 182 1379 AF508761Euplotes aediculatus 1881 67 150 186 1385 AF508756Tetrahymena thermophilaa1753 130 154 177 3760 X54512aEngberg and Nielsen (1990).E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267 261
  5. 5. Unknown FL is also observed not to be associated withany particular ciliate, and like Unknown B, is moder-ately supported [75% (MP), 80% (QPS), and 98% (NJ)]as grouping within a clade containing a number ofgenera (Stylonychia, Gastrostyla, Pleurotricha, Tet-memena, etc.) (Fig. 2). Similar to the macronuclear SSUrDNA gene tree, O. longa and O. granulifera are notobserved to group with each other. In fact, O. granu-lifera is again supported as grouping with Paraurostylaviridis [100% (MP), 52% (QPS), and 100% (NJ)], andO. longa is again observed to group with the UnknownSHS [75% (MP), 94% (QPS), and 94% (NJ) (Fig. 2)], asseen in the SSU rDNA gene analyses (Fig. 1).The corrected sequence divergence of the macronu-clear LSU rDNA gene is higher than that for the mac-ronuclear SSU rDNA gene and ranged from 0.220%[Sterkiella sp. (Aspen) and Sterkiella histriomuscorum]to 30.6% (T. thermophila and E. aediculatus). Similar tothe SSU analysis, E. aediculatus and M. crassus are wellsupported as a solid entity in all phylogenetic treesFig. 1. Strict consensus of 180 most parsimonious trees from analysis of 331 phylogentically informative characters of the nuclear SSU rDNA gene inthe spirotrichous ciliates. The first of the three numbers above internal branches represent bootstrap resampling results (% of 1000 replicates) formaximum parsimony analysis. The second number represents the support based on quartet-puzzling steps (QPS, % of 1000 replicates). The thirdnumber is the bootstrap value using the neighbor-joining algorithm (% of 1000 replicates). An asterisk or branch lacking values had less than 50%bootstrap support.262 E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267
  6. 6. [100% (MP), 82% (QPS), and 99% (NJ)] and are posi-tioned on a long branch in the neighbor-joining tree (notshown) and differ considerably in sequence divergencefrom the remaining ciliates (18.6–23.2%), not includingT. thermophila, from which they differ by 29.4–30.6%.The sequence divergence between O. longa andO. granulifera is considerably higher (10.5%) than thatobserved for the macronuclear SSU rDNA gene. Theunknown taxa were also observed to have higher se-quence divergence values than that noted for the mac-ronuclear SSU rDNA gene. For example UnknownSHS and O. longa differ by 5.3%; Unknown F andP. lanceolata by 1.24%. Unknown B and Unknown FL,which are observed to group with a clade containing anumber of genera, differed from these taxa by 6.16–9.18% and 7.34–10.3%, respectively.3.3. Combined analysis of SSU, LSU, and 5.8S rDNAgenesSequences of the macronuclear SSU, LSU, and 5.8SrDNA genes were combined for phylogenetic analyses.Fig. 2. Strict consensus of 38 most parsimonious trees from analysis of 462 phylogenetically informative characters of the nuclear LSU rDNA gene inthe spirotrichous ciliates. The first of the three numbers above internal branches represent bootstrap resampling results (% of 1000 replicates) formaximum parsimony analysis. The second number represents the support based on quartet-puzzling steps (QPS, % of 1000 replicates). The thirdnumber is the bootstrap value using the neighbor-joining algorithm (% of 1000 replicates). An asterisk or branch lacking values had less than 50%bootstrap support.E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267 263
  7. 7. Parsimony analysis of 838 phylogenetically informativecharacters from the three rDNA genes resulted in eightmost parsimonious trees with a length of 2789 anda consistency index of 0.525, one of which is shown inFig. 3. The topology of the neighbor-joining and quar-tet-puzzling trees were similar and hence only the par-simony tree is shown (Fig. 3). The topology of this tree issimilar to that seen in the previous two analyses, how-ever there is more resolution of the various clades. Forexample, grouping of Unknown F with P. lanceolataand Unknown SHS with O. longa are well supported.Similar to the macronuclear SSU rDNA gene analyses(Fig. 1), Unknown B is moderately supported in a cladewith C. citrina [71% (MP), 84% (QPS), and 70% (NJ)](Fig. 3); however this relationship was not reflected inthe LSU rDNA gene analyses (Fig. 2).3.4. Phylogenetic analysis of the 5.8S rDNA gene andITS regionsPhylogenetic analyses were carried out on 162 nu-cleotides in the 5.8S rDNA, however, phylogenetic treesFig. 3. One of eight most parsimonious trees from combined analysis of 838 phylogenetically informative characters of the nuclear SSU, LSU, and5.8S rDNA genes in the spirotrichous ciliates. The first of the three numbers above internal branches represents bootstrap resampling results (% of1000 replicates) for maximum parsimony analysis. The second number represents the support based on quartet-puzzling steps (QPS, % of 1000replicates). The third number is the bootstrap value using the neighbor-joining algorithm (% of 1000 replicates). An asterisk or branch lacking valueshad less than 50% bootstrap support.264 E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267
  8. 8. showed little or no resolution with the exception ofE. aediculatus and M. crassus, which were well sup-ported as a solid entity by parsimony (100%) andneighbor-joining (100%) bootstrap resampling (notshown). In addition, sequences of the internal tran-scribed spacers (ITS 1 and 2) were also obtained, andphylogenetic trees were constructed (Fig. 4). Alignmentof these sequences was difficult because of the consid-erable variability, and, hence, phylogenetic trees do notinclude M. crassus, E. aediculatus, and T. thermophila.Parsimony analysis of 92 phylogenetically informativecharacters of ITS 1 and 2 resulted in 257 most parsi-monious trees with a length of 252 and a consistencyindex (CI) of 0.600. A strict consensus of these 257 mostparsimonious trees is depicted in Fig. 4; as in the case ofthe previous analyses only the parsimony consensus isshown with bootstrap and QPS values included. The treedepicted in Fig. 4 is similar in topology to the otherfigures (Figs. 1–3) but had less resolution with theexception of a few taxa. For example, Unknown F is wellsupported in grouping with P. lanceolata [100% (MP),82% (QPS), 99% (NJ)], and Unknown SHS, is stronglyassociated with O. longa [100% (MP), 89% (QPS), 100%(NJ)]. These relationships were also noted in the analysesFig. 4. Strict consensus of 257 most parsimonious trees from analysis of 92 phylogenetically informative characters of ITS 1 and 2 of the nuclearrDNA cistron in the spirotrichous ciliates. The first of the three numbers above internal branches represent bootstrap resampling results (% of 1000replicates) for maximum parsimony analysis. The second number represents the support based on quartet-puzzling steps (QPS, % of 1000 replicates.)The third number is the bootstrap value using the neighbor-joining algorithm (% of 1000 replicates). An asterisk or branch lacking values had lessthan 50% bootstrap support.E.A. Hewitt et al. / Molecular Phylogenetics and Evolution 29 (2003) 258–267 265
  9. 9. of SSU, LSU, and combined nuclear rDNA gene anal-yses (Figs. 1–3).Our work expands the seminal report of Bernhardet al. (2001) on phylogenetic relationships among spi-rotrichs based on SSU rDNA to include sequences ofSSU, LSU, 5.8S rDNA and ITS 1 and 2, and we haveextended the analysis to 28 spirotrichs. Nine of theseorganisms were part of the analysis by Bernhard et al.(2001), and our results on phylogenetic relationships arein overall agreement with theirs.The phylogenetic relationships among the 28 spiro-trichs documented by SSU, LSU, 5.8S, and ITS 1 and 2are, in most cases, consistent with classical taxonomybut in other cases, sharply disagree. Classical taxonomyof spirotrichs is based on morphological characteristics,depending particularly on such features as the numberand patterns of cirri and membranelles. Such featuresare quite useful in identifying different organisms, butalone they may be of somewhat limited value for de-fining phylogenetic relationships. Combining classicaltaxonomic criteria with molecular and gene analysesmay ultimately provide the best approximation of phy-logenetic/evolutionary relationships (see for example,Bernhard et al., 2001).In this context the 28 spirotrichs present examples ofvarying contrasts. For example, S. mytilus, S. lemnae,and Stylonychia sp. (Aspen) are morphologically verysimilar; one may be easily mistaken for another whenthe living organisms are viewed microscopically. Thenuclear rDNA sequences agree about their close kin-ship. Similarly, S. histriomuscorum and Sterkiella sp.(Aspen) are virtually indistinguishable morphologically.Their nuclear rDNA sequences reflect a close relation-ship, but show that they are unquestionably differenttaxa. This is affirmed by the micronuclear gene encodingactin I. In S. histriomuscorum the micronuclear actin Igene is divided by nine noncoding IESs (internal elimi-nated segments) into 10 highly scrambled MDSs (mac-ronuclear destined segments), but this gene in Sterkiellasp. (Aspen) is interrupted by eight IESs, creating onlynine MDSs (DuBois and Prescott, 1995). Moreover,although the scrambling patterns of MDSs are strikinglysimilar, corresponding IESs are of different lengths andcontain completely different nucleotide sequences.In contrast to these agreements O. granulifera andO. longa are morphologically similar and therefore havebeen placed in the same genus. Their nuclear rDNAsequences indicate a much more distant phylogeneticrelationship, which is supported by a phylogeneticanalysis of the actin I gene (Croft et al., 2003). Anothermajor contradiction is the position of H. grandinellain the nuclear rDNA tree. By morphological criteriaH. grandinella is placed in an entirely different order,Halteriida (Petz and Foissner, 1992), but the nu-clear rDNA sequences place this organism well withinthe order Stichotrichida with a relatively close kinship toO. granulifera, Paraurostyla viridis, and E. mobilis. Shinet al. (2000) previously found that H. grandinella be-longs to the Oxytrichidae based on SSU rDNA. Ourresults with SSU, LSU, and 5.8S nuclear rDNA are inagreement, and moreover, show the close relationshipbetween H. grandinella and O. granulifera based on SSUrDNA reported by Bernhard et al. (2001). Further, theamino acid sequence of actin I also places H. grandinellasolidly within the order Stichotrichida (Croft et al.,2003).The rDNA tree suggests that U. pisces and U. gallinamay not belong to the same genus. Oxytricha sp. (Misty)probably belongs to the family Oxytrichidae (generaGastrostyla, Sterkiella, Oxytricha, Pleurotricha, Styl-onychia, and Tetmemena), but it is distantly separatedboth from O. longa and O. granulifera. Holostichapolystylata has been placed in the family Holostichidaeand U. grandis in the family Urostylidae (Lynn andCorliss, 1991), but the nuclear rDNA analysis indicatesa close phylogenetic relationship between these two or-ganisms.Among the 28 organisms represented in the nuclearrDNA tree four are as yet unidentified organisms, Un-known F, Unknown B, Unknown SHS, and UnknownFL. We prefer at this juncture to refer to these organ-isms by the accession numbers for their nuclear rDNA.Unknown F may belong to the genus Pleurotricha andUnknown SHS may share the same genus with O. longa.The rDNA tree does not suggest possible genus assign-ments for Unknown B and Unknown FL to any of thegenera represented in this analysis.AcknowledgmentsThis work is supportedby NIGMS Research Grant#R01GM 56161 and NSF Research Grant MCB-9974680 to D.M. Prescott. K.M. M€uuller is supported bystart up funds from the University of Waterloo. D.J.Hogan was partially supported by the Hughes Under-graduate Biological Science Education Initiative, theUniversity of Colorado Undergraduate Research Op-portunities Program, and the University of ColoradoCancer Center. Robin Gutell was supported by NIHR01 GM 48207. We are grateful to Gayle Prescott forpreparation of the manuscript. 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